Investigating the Role for IL-21 in Rabies Virus Vaccine-induced Immunity

Over two-thirds of the world''s population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of T_fh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.     

Protective Vaccine-Induced CD4+ T Cell-Independent B Cell Responses against Rabies Infection

A major goal in rabies virus (RV) research is to develop a single-dose postexposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with rabies prevention in humans, and save lives. Live replication-deficient RV-based vaccines are emerging as promising single-dose vaccines to replace currently licensed inactivated RV-based vaccines. Nonetheless, little is known about how effective B cells develop in response to live RV-based vaccination. Understanding this fundamental property of rabies immunology may help in developing a single-dose RV vaccine. Typically, vaccines induce B cells secreting high-affinity, class-switched antibodies during germinal center (GC) reactions; however, there is a lag time between vaccination and the generation of GC B cells. In this report, we show that RV-specific antibodies are detected in mice immunized with live but not inactivated RV-based vaccines before B cells displaying a GC B cell phenotype (B220⁺GL7^hiCD95^hi) are formed, indicating a potential role for T cell-independent and early extrafollicular T cell-dependent antibody responses in the protection against RV infection. Using two mouse models of CD4⁺ T cell deficiency, we show that B cells secreting virus-neutralizing antibodies (VNAs) are induced via T cell-independent mechanisms within 4 days postimmunization with a replication-deficient RV-based vaccine. Importantly, mice that are completely devoid of T cells (B6.129P2-Tcrβ^tm1Mom Tcrδ^tm1Mom/J) show protection against pathogenic challenge shortly after immunization with a live replication-deficient RV-based vaccine. We show that vaccines that can exploit early pathways of B cell activation and development may hold the key for the development of a single-dose RV vaccine wherein the rapid induction of VNA is critical.     

A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD₅₀) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10⁶ PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10⁸ PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10⁸ PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.     

Rabies Virus Infection Induces Type I Interferon Production in an IPS-1 Dependent Manner While Dendritic Cell Activation Relies on IFNAR Signaling

As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5−/− and RIG-I−/− mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I−/− cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1−/− mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.     

Pan-HSV-2 IgG Antibody in Vaccinated Mice and Guinea Pigs Correlates with Protection against Herpes Simplex Virus 2

We lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2''s 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2''s antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 ⁻ viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.     

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m^−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m^−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8⁺ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.     

Protective Role of Gamma Interferon during the Recall Response to Influenza Virus

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4⁺ and CD8⁺ T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.     

A mouse model of the pathogenesis and postexposure prophylaxis of rabies

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.     

Reduced protection of RIPK3-deficient mice against influenza by matrix protein 2 ectodomain targeted active and passive vaccination strategies

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3^−/− mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3^−/− mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3^−/− vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3^−/− mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3^−/− mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3^−/− mice. Likewise following IAV infection of Ripk3^−/− mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3^−/− mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3^−/− mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.Subject terms: Infection, Viral infection

     

Immunization of Knock-Out α/β Interferon Receptor Mice against High Lethal Dose of Crimean-Congo Hemorrhagic Fever Virus with a Cell Culture Based Vaccine

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The disease has been reported in many countries of Africa, Asia, the Middle East, and in Eurasia. During the past decade, new foci of CCHF have emerged in the Balkan Peninsula, southwest Russia, the Middle East, western China, India, Africa, and Turkey. CCHF virus produces severe hemorrhagic manifestations in humans with fatality rates up to 30%. Vaccine development efforts have been significantly hampered by a lack of animal models and therefore, no protective vaccine has been achieved. Lately, IFN α/β receptor deficient (IFNAR^−/−) mice have been established as a novel small animal model of CCHF virus infection. In the present study, we found that IFNAR^−/− mice highly susceptible to CCHF virus Turkey-Kelkit06 strain. Immunization with the cell culture based vaccine elicited a significant level of protection against high dose challenge (1,000 PPFU) with a homologous CCHF virus in IFNAR^−/− mice.     

Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta-propiolactone-inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.     

Intracerebral administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus

Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.     

Ns1 Is a Key Protein in the Vaccine Composition to Protect Ifnar(?/?) Mice against Infection with Multiple Serotypes of African Horse Sickness Virus

African horse sickness virus (AHSV) belongs to the genus Orbivirus. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2 and NS1 proteins from AHSV-4. IFNAR^(−/−) mice inoculated with DNA/rMVA-VP2,-NS1 from AHSV-4 in an heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies specific of AHSV-4. In addition, vaccination stimulated specific T cell responses against the virus. The vaccine elicited partial protection against an homologous AHSV-4 infection and induced cross-protection against the heterologous AHSV-9. Similarly, IFNAR^(−/−) mice vaccinated with an homologous prime-boost strategy with rMVA-VP2-NS1 from AHSV-4 developed neutralizing antibodies and protective immunity against AHSV-4. Furthermore, the levels of immunity were very high since none of vaccinated animals presented viraemia when they were challenged against the homologous AHSV-4 and very low levels when they were challenged against the heterologous virus AHSV-9. These data suggest that the immunization with rMVA/rMVA was more efficient in protection against a virulent challenge with AHSV-4 and both strategies, DNA/rMVA and rMVA/rMVA, protected against the infection with AHSV-9. The inclusion of the protein NS1 in the vaccine formulations targeting AHSV generates promising multiserotype vaccines.     

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3^−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.     

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.     

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28^−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28^−/loxCre^+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28^−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28^−/loxCre^+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28^−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T_FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28^−/− mice and tamoxifen treated CD28^−/loxCre^+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28^−/loxCre^+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.     

Overexpression of Interleukin-15 Compromises CD4-Dependent Adaptive Immune Responses against Herpes Simplex Virus 2

Interleukin-15 (IL-15) is necessary for the development and function of NK/NKT cells and the maintenance of naive and memory CD8⁺ T cells. In the absence of IL-15, protective innate immunity is not available; however, a functional adaptive immune response against vaginal herpes simplex virus 2 (HSV-2) is generated. Mice overexpressing IL-15 (IL-15tg mice) have higher numbers of NK cells, greater NK-derived gamma interferon, and more CD8⁺ T cells. Here we examined the consequences of IL-15 overexpression for innate and adaptive immunity against genital HSV-2. Surprisingly, IL-15tg mice immunized against HSV-2 were not protected against genital HSV-2 challenge compared to control immunized mice. IL-15tg mice had a higher frequency of NK cells in the genital mucosa than control mice. However, immunized IL-15tg mice had significantly lower numbers of HSV-2-specific CD4⁺ T cells than B6 mice. We then confirmed that CD4⁺ T cells, but not CD8⁺ T cells, are essential for protection against intravaginal HSV-2 challenge. Since we observed less protection in immunized IL-15tg mice, we then examined if the adaptive immune responses generated in an environment with overexpression of IL-15 could provide protection against HSV-2 in an environment with normal levels of IL-15 expression. We adoptively transferred immunized cells from IL-15tg and B6 mice into naive RAG-1^−/− mice and found that the cells from immunized IL-15tg mice were able to provide protection in this IL-15-normal environment. Our data suggest that overexpression of IL-15 results in a reduced CD4⁺ T cell-mediated adaptive immune response against genital HSV-2.     

B Cell Infection and Activation by Rabies Virus-Based Vaccines

Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4⁺ T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4⁺ OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical.     

Herpes Simplex Virus 2 ICP0? Mutant Viruses Are Avirulent and Immunogenic: Implications for a Genital Herpes Vaccine

Herpes simplex virus 1 (HSV-1) ICP0 ⁻ mutants are interferon-sensitive, avirulent, and elicit protective immunity against HSV-1 (Virol J, 2006, 3:44). If an ICP0 ⁻ mutant of herpes simplex virus 2 (HSV-2) exhibited similar properties, such a virus might be used to vaccinate against genital herpes. The current study was initiated to explore this possibility. Several HSV-2 ICP0 ⁻ mutant viruses were constructed and evaluated in terms of three parameters: i. interferon-sensitivity; ii. virulence in mice; and iii. capacity to elicit protective immunity against HSV-2. One ICP0 ⁻ mutant virus in particular, HSV-2 0ΔNLS, achieved an optimal balance between avirulence and immunogenicity. HSV-2 0ΔNLS was interferon-sensitive in cultured cells. HSV-2 0ΔNLS replicated to low levels in the eyes of inoculated mice, but was rapidly repressed by an innate, Stat 1-dependent host immune response. HSV-2 0ΔNLS failed to spread from sites of inoculation, and hence produced only inapparent infections. Mice inoculated with HSV-2 0ΔNLS consistently mounted an HSV-specific IgG antibody response, and were consistently protected against lethal challenge with wild-type HSV-2. Based on their avirulence and immunogenicity, we propose that HSV-2 ICP0 ⁻ mutant viruses merit consideration for their potential to prevent the spread of HSV-2 and genital herpes.